Fluorescent aminoglycoside antibiotics and methods for

accurately monitoring uptake by bacteria

Mahnaz Sabeti Azad, Maho Okuda, Mélina Cyrenne, Mickael Bourge,
Marie-Pierre Heck, Satoko Yoshizawa, Dominique Fourmy

To cite this version:

Mahnaz Sabeti Azad, Maho Okuda, Mélina Cyrenne, Mickael Bourge, Marie-Pierre Heck, et al..
Fluorescent aminoglycoside antibiotics and methods for accurately monitoring uptake by bacteria.
ACS Infectious Diseases, American Chemical Society, 2020, 6 (5), pp.1008-1017. �10.1021/acsin-
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Fluorescent aminoglycoside antibiotics and methods for accurately
monitoring uptake by bacteria
Mahnaz Sabeti Azad†, Maho Okuda†,‡, Mélina Cyrenne†, Mickael Bourge†, Marie-Pierre Heck§, Satoko
Yoshizawa†, and Dominique Fourmy*,†.
†
Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), 91198, Gif-sur-Yvette, France.
§
Université Paris-Saclay, CEA, Service de Chimie Bio-organique et de Marquage, 91191 Gif-sur-Yvette, France.
Email: dominique.fourmy@i2bc.paris-saclay.fr
Characterizing how multidrug-resistant bacteria circumvent the action of clinically used or novel antibiotics requires a detailed
understanding of how the antibiotics interact with and cross bacterial membranes to accumulate in the cells and exert their action.
When monitoring the interactions of drugs with bacteria it remains challenging to differentiate functionally relevant internalized
drug levels from non-specific binding. Fluorescence is a method of choice for observing dynamics of biomolecules. In order to
facilitate studies involving aminoglycoside antibiotics, we have generated fluorescently labeled aminoglycoside derivatives with
uptake and bactericidal activities similar, albeit with a moderate loss, to those of the parent drug. The method combines fluores-
cence microscopy with fluorescence-activated cell sorting (FACS) using neomycin coupled to non-permeable cyanine dyes. Fluo-
rescence imaging allowed membrane-bound antibiotic to be distinguished from molecules in the cytoplasm. Patterns of uptake were
assigned to different populations in the FACS analysis. Our study illustrates how fluorescent derivatives of an aminoglycoside
enable a robust characterization of the three components of uptake: membrane binding, EDPI, and EDPII. Because EDPI levels are
weak compared to the two other types of accumulation and critical for the action of these drugs, the three components of uptake
must be taken into account separately when drawing conclusions about aminoglycoside function.

Keywords: Fluorescence; antibiotics; aminoglycosides; uptake; flow cytometry; microscopy.

Aminoglycosides are among the oldest antibiotics in clinical
use. These broad spectrum antibiotics are used against clini-
cally important bacteria and for treatment of infections
caused by multidrug-resistant bacteria.1,2 In the past decade,
novel aminoglycosides have been developed with decreased
toxicity and susceptibility to resistance.2–7 Many widely used
aminoglycosides contain a deoxystreptamine ring (2-DOS)
that is essential for antimicrobial activity. The 2-DOS ami-
noglycosides bind both subunits of the ribosome and inhibit
protein synthesis.8–10 These drugs cause miscoding 11 via a
direct interaction with ribosomal RNA (rRNA) of the small
subunit.10,12–16 Some 2-DOS aminoglycosides also interact at
a specific site within the large ribosomal subunit resulting in
inhibition of translocation 17,18 or recycling.19 In addition,
aminoglycosides have been reported to inhibit initiation 20,21
and perturb ribosome biogenesis.22–24
Aminoglycoside uptake occurs through two energy-
dependent phases known as EDPI and EDPII.25–27 EDPI is
known to be dependent on the proton motive force (PMF).28–
30
EDPII requires active protein synthesis by aminoglyco-
side-sensitive ribosomes and, therefore, is energy dependent.
The lethal mode of action of aminoglycosides is thought to
occur through induction of ribosomal miscoding, which
produces misfolded proteins that when inserted into the inner
membrane trigger an increase of permeability and conse-
quently a diffusive entry of the drug in EDPII.31 Increase in
cell killing efficiency is interpreted as an increase in amino-
glycoside uptake. Measuring aminoglycoside uptake is im-
portant for understanding the basis of aminoglycoside action.
This has been often achieved by study of cultured cells treat-
ed with radioactively labeled aminoglycosides.25,26,32 A
method has been reported for measuring intracellular levels
of kanamycin that relies on an ELISA, but it requires cell
lysis.33 Novel tools making use of fluorescent antibiotics are
emerging that enable characterization of the interactions of
antibiotics with bacteria at the level of organelles or even
whole organisms.34 For example, gentamicin and tobramycin
coupled to Texas Red have been widely used in this type of
analysis; however this dye, by itself, penetrates cells, com-
plicating analysis.35 Recently developed techniques make use
of spectrofluorimetry, microspectrofluorimetry and kinetics
microspectrofluorimetry for single-cell analysis of naturally
fluorescent antibiotics or fluorescently-labeled com-
pounds.36,37 In addition, mass spectrometry was combined
with spectrofluorimetry to provide insights into the efflux
mechanisms of fluoroquinolones.38
Depending on the analysis, the membrane-bound fraction of
a drug may be quantified, biasing the measurement of true
internalized drug levels. For aminoglycosides, it is also im-
portant to differentiate EDPI and EDPII phases, as only
EDPI leads to cell death. EDPII is a consequence of internal-
ization of the drug through the membrane damaged due to
EDPI. In addition, because each cell may respond differently
to antibiotics, an accurate description of drug uptake requires
single-cell analysis. To finely evaluate aminoglycoside up-
take, we developed a series of cyanine-modified neomycin
 Neomycin Neo-Cy5 hydrazone (CCCP) on Neo-Cy5-induced bacterial cell death.
P. aeruginosa PA14 3.25 150 Addition of CCCP protected cells from the drug-induced
killing (Figure 3 and Figure S8), demonstrating that, as for
Measured in minimal medium MOPS-G. other aminoglycosides, the accumulation of Neo-Cy5 is PMF
Neo-Cy5 causes misreading during ribosomal decoding. dependent.
Aminoglycosides cause miscoding in vitro at concentrations 4 **** ****
in the range of 10-100 µM.5,11,43,44 Since Neo-Cy5 binds the Untreated

log 10 (survival percentage)

ribosomal decoding site on the small subunit (Figure 2(a)), 2
CCCP 30 µM
we tested the capacity of Neo-Cy5 to induce misreading
using a dual-luciferase gain-of-function assay. In this assay, 0

misreading restores the enzymatic activity of a mutant firefly

luciferase. In the mutant, codon 245, which in the wild-type -2

mRNA is CAC (coding for histidine), is replaced by the

near-cognate CGC codon (arginine).5 Translation activity -4

was analyzed by comparison to the signal due to wild-type

-6
Renilla luciferase, which served as internal standard. Like
neomycin, Neo-Cy5 caused miscoding however effects were

tic

µM
µM
io

48
1
tib
attenuated by more than one order of magnitude (Figure

in

y5
an

yc

-C
m
o
2(b)).

eo
N

eo

N
N
a) Figure 3. Neo-Cy5-induced cell killing is PMF dependent.
Neomycin
Neomycin Neomycin
Neo-Cy5 Cy5
Survival of E. coli MG1655 cells after a 4.5-h treatment with 1.5
µM neomycin or 48 µM Neo-Cy5 with and without a 2-h pre-
0.05 µM

0.05 µM

incubation with 30 µM CCCP. Plotted are values relative to

0.2 µM

0.2 µM
0.5 µM
control

0.1 µM

0.1 µM
1 µM

1 µM
5 µM

untreated control culture. Error bars are s. e. m. and were calcu-

A C G U
0

lated from three independent experiments. Significant differ-

ences, calculated by the Mann–Whitney test, are indicated (****
A1408 P < 0.0001).

G1405
A1408 b) FACS and TIRF microscopy reveal drug peripheral
Fig
(F-luc mutant) / (F-luc wt x 10-5)

accumulation and strong interaction with membranes.

21000 Neo-Cy5
Because cationic aminoglycosides can interact with the
Neomycin membranes of the bacterial cell surface, we devised a proto-
18000
col to measure only tightly-bound or internalized Neo-Cy5.
15000 We first removed weakly membrane-bound and not internal-
ized molecules by washing the cells on mixed cellulose ester
12000
filters. At least three washes with MOPS-G at 37 °C were
9000 necessary to remove non-internalized drug (Figure S9). We
measured Neo-Cy5 uptake by E. coli MG1655 cells in the
6000 exponential phase of growth in the presence of Neo-Cy5 at a
3000
concentration three times the MIC. After filtration, cells
were recovered and immediately analyzed by FACS. After a
0 short 5-minute exposure to Neo-Cy5, a signal due to the
0,01 .
0,1 1 10 100 conjugate was clearly distinguished from the background
An6bio6cs (µM) fluorescence (Figure 4(a)). We verified that the signal was
dependent on the neomycin moiety: TIRF microscopy
Figure 2. Neo-Cy5 binds ribosomal A site and induces mis- showed that the Cy5 dye alone was not incorporated into live
reading. a) Binding of neomycin (left) and Neo-Cy5 (right) to Figure 2
bacterial cells and that Cy5 did not interact strongly with
ribosomes was assayed using DMS chemical probing. Left lane membranes (Figure S10 and Figure 4(a)). Because the ob-
is a control reaction with no DMS added. Right lanes are reac-
served signal for Neo-Cy5 could correspond to molecules
tions in the presence of increasing concentrations of neomycin
tightly bound to the membrane or internalized in the
or Neo-Cy5. The protected nucleotide A1408 that belongs to the
periplasm or cytoplasm, we inspected the localization of the
neomycin binding site (Figure 1(b) is marked with an arrow. b)
Mis-incorporation of amino acids was assayed using a gain-of- drug by TIRF microscopy (Figure 4(b)). Here, periplasmic
function assay. Wild-type and mutant firefly luciferase reporters accumulation would correspond to the self-promoted mecha-
were used for in vitro translation reactions together with the nism previously proposed where aminoglycoside cross irre-
Renilla luciferase, which served as an internal control. Error versibly the outer membrane via the lipopolysaccharides in
bars are s. e. m. for three independent experiments. E. coli.45 After a 5-minute incubation, Neo-Cy5 accumulated
at the periphery of the cell and did not overlap with the fluo-
rescence of Spinach-tagged ribosomes46 (Figure 4(c) and
Neo-Cy5 has proton motive force-dependent bactericidal Figure S11)). We conclude that Neo-Cy5 bound at the mem-
activity. Aminoglycoside accumulation in bacterial cells is brane of a cell or internalized in the periplasm can readily be
known to be dependent on PMF.28 We therefore tested the
effect of the protonophore carbonyl cyanide m-chlorophenyl
 derivatives that have antibacterial activity similar to the
parent aminoglycoside. When used with adequate protocols,
these derivatives allow single-cell analysis of aminoglyco-
side uptake by fluorescence microscopy and fluorescence-
activated cell sorting (FACS).
RESULTS AND DISCUSSION
Generation of Neo-cyanine derivatives. Radioactivity
based assays25,26,32 and ELISA33 do not report on accumula-
tion at the single cell level. A fluorescent derivative with
preserved uptake and bactericidal activities would facilitate
analysis of uptake with single-cell resolution. We previously
showed that the 5” position of neomycin could be modified
with various functional groups without compromising anti-
microbial activity (Figure S1). Analysis of the structures of a
neomycin-ribosome complex indicated that functional
groups introduced at this position are accommodated in the
major groove of helix 44 of the 16S rRNA in a conformation
that does not interfere with drug binding or induction of
miscoding.
We used a derivative of neomycin where the hydroxyl group
at the 5” position was substituted with S-(CH2)2-NH2 to
allow introduction of extra chemical groups by coupling
(Supporting information, product 5).39 Addition of a
thyminylacetamidoethylthio or uracil (Neo-U) at the 5”
position preserved the antimicrobial activity against E. coli
as measured using an MIC assay (Figure S1 and Table S1).
As expected, Neo-U bound to the A site on the small ribo-
somal subunit, although affinity was slightly decreased com-
pared to the unmodified neomycin (Figure S2). This result
indicated that large chemical modifications can be intro-
duced at the 5” position of ring III without strongly perturb-
ing A-site binding or antimicrobial properties.
We next labeled neomycin with sulfonated cyanines Cy3 and
Cy5 (Figures S3,S4,S5 and S6), which are non-membrane
permeable dyes suitable for fluorescence microscopy.40 The
spectral properties of Neo-Cy5 conjugate (Figure 1(a)) do
not differ from those of the free dye (Figure S7). We verified
that the fluorescent probes are accommodated without any
steric clash by docking into the crystal structure of E. coli
ribosomes bound to neomycin (PDB code 4v52). The Cy3
and Cy5 probes, which are bulkier than the uracil base, likely
lie in a cavity between helices h27 and h44 of 16S ribosomal
RNA (Figure 1(b)).
es : a) b) 30S 50S
Figure 1. Structure of the Neo-Cy5 conjugate and accommoda-
tion at the ribosomal A site. a) Structure of Neo-Cy5 in which
Cy5 is coupled to ring III of neomycin. b) Neomycin binding
sites in h44 of the small ribosomal subunit (blue) and in H69 of
the large ribosomal subunit (beige) (PDB code 4v52). Small and
large ribosomal proteins are shown in purple and green, respec-
tively. The red box indicates a cavity near the neomycin binding
site in h44 where Cy5 can be accommodated. Nucleotide A1408
that is part of the neomycin binding site is indicated.
Neo-cyanine derivatives are active antibiotics. Next, we
verified that Neo-Cy5 binds the decoding site of 16S rRNA
in 70S ribosomes. Footprinting analysis of ribosomal parti-
cles bound to Neo-Cy5 showed that the conjugate interacts
with the decoding site of the small ribosomal subunit in a
manner similar to neomycin and Neo-U, and like Neo-U,
Neo-Cy5 has slightly lower affinity than the parent antibiotic
(Figure 2(a)). We next tested the antimicrobial activity of
Neo-Cy5. The measured MIC in minimal medium was
slightly higher than that of neomycin (Table 1) and similar to
the MICs for Neo-U (Table S1) and closely related amino-
glycosides.41,42 The MIC of Neo-Cy3 was also similar to that
of Neo-Cy5 (Table 1).
MIC values were also measured in Mueller-Hinton broth for
Gram negative and positive bacteria including ESKAPE
pathogens (Table 2). Tested strains included clinically rele-
vant Gram negative pathogens such as Pseudomonas aeru-
ginosa, A. baumannii, K. pneumoniae, S. typhimurium, as
well as the Gram positive S. aureus USA300. MIC values for
the parental drug neomycin were higher in rich medium
when compared to minimal medium. This effect was strong-
er for the Neo-Cy5 conjugate as MIC values were increased
by factors ranging from 20 to 40 compared to the values
measured for neomycin (Table 2). The MIC value was not
measurable for Pseudomonas aeruginosa in rich medium and
found to be 150 µg/ml in MOPS-G (Table 3).
Table 1. MIC values in µg/ml of neomycin-cyanine deriv-
ative conjugates against E. coli MG1655.
Neomycin Neo-Cy5 Neo-Cy3
M9 medium 3.0 13 ND
MOPS medium 1.1 10.4 5.2
Measured in minimal medium MOPS-G. ND: not determined.
Table 2. MIC values of Neo-Cy5 in µg/ml against Gram
negative and positive strains.

Neomycin Neo-Cy5
E. coli MG1655 2.5 75
neomycin
P. aeruginosa PA14 2.5 > 150

Neo-Cy5Neo-Cy5
A. baumannii DSM30011 5.0 75
K. pneumoniae LM21 1.25 25
NHS Cy5 S. typhimurium14028 1.25 50
NHS Cy5 h 27
B. subtilis 0.25 6.25
* H 69 M. luteus 1.5 6.25
ation of Neo-Cy5. The primary amine group that reacts A1408
S. aureus USA300 2.5 75
ighlighted by the red circle. Other amino groups are
Neo-Cy5 h 44
foxycarbonyl
Neo-Cy5.groups.
The primary amine group that reacts Position Cy5
Measured in cation-adjusted Mueller-Hinton broth.
ted by the red circle. Other amino groups are S12
bonyl groups. Table 3. MIC values of Neo-Cy5 in µg/ml against P. aeru-
Figure 1
ginosa PA14.
detected by FACS and visualized by TIRF fluorescence
microscopy.
Neo-Cy5 binding to bacterial membranes is saturable.
Binding of Neo-Cy5 to the bacterial membranes was found
to be instantaneous as visualized in real time by FACS (Fig-
ure S12). Membranes of E. coli showed saturability when
exposed for 5 minutes to increasing concentrations of Neo-
Cy5 (Figure 4(d,e)). At low concentrations, there was strong
heterogeneity in fluorescence levels of individual cells (Fig-
ure 4(d)). At the highest concentrations, even though the
exposure time was short to ensure that only the drug interac-
tion with the membrane was monitored, in a fraction of cells
high levels of uptake was observed (Figure 4(d)). Experi-
ments described in the next section demonstrated that this
fraction corresponds to cells with cytoplasmic uptake. In
cells treated with 64 µM of Neo-Cy5, 2.9% of cells had
detectable levels of Neo-Cy5 in the cytoplasm. This value is
above the known fraction of 0.3% of cells that are dead in a
fast growing bacterial population.47 Therefore there is rapid
cytoplasmic uptake at high concentrations of Neo-Cy5.
Neo-Cy5 is internalized mainly through EDPII. E. coli
MG1655 cells were treated with 32 µM Neo-Cy5 for 30
minutes, the time period corresponding to the onset of ami-
noglycoside-induced exponential killing.48 When cells were
imaged using TIRF, we observed that fluorescence signals
were distributed over the entire cell volume for most of the
cells (time 30 min 88%, n=57), (Figure 5(a)). This clearly
indicated cytoplasmic uptake. Most of the cells had low
levels of fluorescence corresponding to EDPI uptake. A low
percentage of cells had bright Cy5 fluorescence signals sug-
gestive of EDPII accumulation. For 60 min of incubation,
100% of the cells had cytoplasmic uptake (n=119). Cells
exposed to Neo-Cy5 overnight showed morphological de-
fects (Figure S13) similar to what has been previously de-
scribed for cells treated with aminoglycosides,49,50 in particu-
lar foci formation at poles.
Addition of the protein synthesis inhibitor chloramphenicol
prior to exposure to aminoglycosides decreases EDPI and
abolishes EDPII.51 We pre-incubated the cells with chloram-
phenicol before the addition of a lethal concentration of Neo-
Cy5 to evaluate the contributions of EDPI and EDPII uptake
by FACS (Figure 5(b)). Differences in profiles obtained with
or without chloramphenicol were very minor at 30 minutes;
TIRF imaging showed that EDPI already occurred for most
of the cells (Figure 5(a)). Only a slight shift of the distribu-
tion toward stronger levels of fluorescence was observed
with time. This result revealed that EDPI uptake is indeed
difficult to capture by FACS. The shift of the distribution in
absence of chloramphenicol was more pronounced at 60 and
150 minutes in agreement with an EDPII-driven cytoplasmic
uptake. We concluded that little Neo-Cy5 accumulates in the
cytoplasm via EDPI when compared to membrane-bound or
cytoplasmic levels that resulted from the EDPII-driven pro-
cess.
Accumulation of Neo-Cy5 and loss of cell viability. We
next performed a detailed kinetic analysis of Neo-Cy5 up-
take at the single-cell level. Cell viability was monitored in
parallel. Like observed for other aminoglycosides48, Neo-
Cy5 displayed the characteristic lag followed by exponential
killing of the cells (Figure 5(c)). The plot of the sum of the
fluorescence levels as a function of time calculated from our
 100
a) Bacteria alone
b)
75
Cy5
Neo-Cy5
50
Counts

25

0
0 103 104 105 106 107
Cy5
c) BF
Cy5 +
Spinach
d) e)
0 %
cytoplasmic

0.4

1
200
Fluorescence intensity (a. u.)

spinach
160 5 0.08 %
neo-cy5

120 10 0.04 %

80 0.03 %
16
40
32 0.49 %
Counts

0
0 1 2 3 64 2.9 %
Distance (µm)
102 103 104 105
Cy5
Figure 4. Fluorescence microscopy and FACS enable detection of Neo-Cy5 bound to E. coli MG1655 cells. a) Figure 4 submitted
FACS analysis of cells
exposed to 32 µM Cy5, 32 µM Neo-Cy5, or no dye for 5 min at 37 °C. Cells were washed on a 0.45 µm-pore-size HAWP membrane filter.
Binding of Neo-Cy5 was clearly distinct from cellular autofluorescence and residual binding of the free dye. b) Representative brightfield
and TIRF microscopy images of cells incubated for 5 min with Neo-Cy5 as in panel “a”. Scale bar: 5 µm c) Neo-Cy5 does not localize
with ribosomes after a 5-minute incubation. E. coli cells expressing Spinach-tagged ribosomes were exposed to Neo-Cy5 and imaged. Left:
Brightfield. Right: Neo-Cy5 (red), Spinach (green). Bottom: plot of a cross section of fluorescence signal for a representative cell with the
peripheral pattern. d) Saturability of bacterial membranes to Neo-Cy5 binding. Cells were incubated for 5 min at 37 °C with increasing
concentrations of Neo-Cy5, filtered, and analyzed by FACS. The percentage of cells with cytoplasmic uptake was calculated from the
number of cell counts with a Cy5 signal higher than the red threshold line divided by the total number of cell counts. e) Binding curve of
the membrane-bound Neo-Cy5.
single-cell analysis recapitulated previous data obtained on the membrane-bound fraction (ratio of medians 120 min/5
cell ensembles.26 During the first 20 minutes of incubation min) (Figure 5(c)). Finally, at the beginning of the transition
with the drug, fluorescence levels increased slightly while time point (40 minutes), the cytoplasmic drug levels that
cell survival remained intact. During this period, Neo-Cy5 accumulated during EDPI, accounted for approximately one
localized at the cell periphery (Supplementary Figure 13). At third of the total levels, the other two thirds corresponded to
40 minutes, the cytoplasmic pattern became dominant with membrane/periplasmic interaction or accumulation (Figure
stronger drug accumulation (Figure 5(c,d) and Figure S14) 5(c)).
concomitantly with the onset of the decrease in cell survival
rate (Figure 5(c)). This period corresponded to a transition
CONCLUSIONS
between EDPI and EDPII. The onset of EDPII was previous-
ly considered to be the first time point at which uptake be- With the purpose of improving methodologies for measuring
comes linear and rapid.26 This time point for Neo-Cy5 was aminoglycoside uptake by bacteria we used click chemistry
around 50 minutes (Figure 5(c)). During the 20-minute tran- to develop fluorescent conjugates of neomycin that have
sition period (40 to 60-min) 90% of the cells lost viability uptake and bactericidal properties similar to the ones of the
with a sharp increase in drug uptake. After 80 minutes, 99% parent aminoglycoside. We coupled sulfonated cyanine dyes
of the cells were dead and levels of accumulation continued that are not membrane-permeable to neomycin allowing
to increase steeply and linearly during 40 minutes, reaching a clear visualization of the uptake. The obtained Neo-Cy3 and
factor of approximately 10 of increase taking as a reference Neo-Cy5 derivatives exhibited uptake properties strictly
a) a)a) 1
1
b)b)100
b) 100 2 min
2 min

50 50
2 2
1 0
1 100 0
30 min
100 30 min
2 EDPI
3 50
2 EDPI
2 min 3 3 50
0
100 60 min
2 min 3
0 EDPII
1 50 100 60 min
1
1
0
100 50
150 min
EDPII
Neo-Cy5

2
1 Neo-Cy5 + Cm
50
0
100 150 min
2 0

Cell counts
102 103 104 105 Neo-Cy5

30 min 2 50 Cy5 Neo-Cy5 + Cm

1
2 0
102 103 104 105
1
30 min 2
Cy5
9 3
2 1 c) 8
EDPI EDPII

n= 240

Cy5 intensity x105

n=
3
1 CFU Log 10 7
2
60 min 3
2
3 d) 6

5
Figure 5 submitted
1
2 n= 149
n= 86
n= 240 4 0
Cy5 intensity x105

Cy5 intensity

n= 217 n= 110 n= 216

3
0 50 100
2 60 min 3
n= 174 Time (min)

Figure 5 submitted
2
Cy5 intensity 104

1 EDPI 0.35

n= 190
1
Membrane 0.65

0 0
0 20 40
Time (min)
Time (min)
Figure 5. EDPI-mediated uptake can be distinguished from EDPII-mediated uptake. a) Representative brightfield and TIRF microscopy
images of cells after 2, 30 and 60 min of exposure to 32 µM Neo-Cy5. Scale bar: 5 µm b) E. coli cells treated with 32 µM Neo-Cy5 with or
without 25 µg/mL of chloramphenicol were incubated for the times indicated. At each time point, cells were filtered, washed, and analyzed
by FACS. Cell counts were normalized to 100%. Weak cytoplasmic uptake, which corresponded to EDPI was detected as an upper shift of
the blue population compared to the red population. The stronger shift corresponding to EDPII levels of uptake is highlighted by an arrow.
c) Killing curve obtained for E. coli with monitoring of Neo-Cy5 uptake. Cells were grown exponentially at 37°C in MOPS medium con-
taining glucose to an OD600 of 0.2, diluted 10 times in fresh medium before addition of Neo-Cy5. At different time points, cells were col-
lected and filtered to remove drug excess. Cells were plated on agar pads for fluorescence microscopy (Figure S14) and fluorescence quan-
tification to generate panel d and serial dilutions were made and plated on LB agar for c.f.u. counting. The kinetic of Neo-Cy5 uptake on
the cell population is represented by the sum of the single-cell analysis of fluorescence signals displayed in panel d. The rapid and linear
EDPII phase is highlighted by a dashed line. d) Single-cell quantification of Neo-Cy5 accumulation as a function of time. Red lines high-
light the medians, boxes show 75/25%, and vertical dashed lines indicate 90/10% of the maxima.
dependent on the neomycin moiety demonstrating that this
type of conjugate can be widely used to report on aminogly-
coside uptake. In addition, these dyes have spectral proper-
ties that make them suitable for fluorescence microscopy.
The Neo-Cy5 conjugate is active against Gram-positive and
negative strains with the exception of Pseudomonas aeru-
ginosa in rich medium. This pathogen is known to resist to
multiple drugs owing to a membrane of low permeability,
the expression of multidrug efflux pumps and its adaptation
to the presence of antibiotics. The efflux pump MexX-
MexY-OprM plays a role in intrinsic resistance to aminogly-
cosides.52–54 The pump has moderate drug specificity as
aminoglycosides, macrolides and tetracycline are sub-
strates.55 Structural data obtained for this type of pumps
provided details on their architecture and revealed indeed
multiple binding pockets for various drugs.56–58 It remains to
be investigated if the presence of the fluorescent dye will
affect the efflux mechanism in Pseudomonas aeruginosa.
Using Neo-Cy5 conjugate, we devised a method for accurate
monitoring of aminoglycoside uptake. Membrane binding of
Neo-Cy5 was found to account for a large fraction of the
fluorescence signal. We showed that we could remove the
excess of drug and the weakly bound molecules, which oth-
erwise masked the real levels of uptake, by filtration and
washing. Using TIRF microscopy we ensured the validity of
FACS observations. Using FACS, we monitored drug bind-
ing and intracellular accumulation. Neo-sulfonated-cyanine
derivatives did not suffer from dye permeability drawbacks
observed for dyes such as Texas Red.35
Aminoglycosides are bound to the bacterial cell membrane
and accumulate in the cytoplasm of bacterial cells through
EDPI and EDPII. The membrane-bound and EDPII compo-
nents accounted for a large fraction of the total levels of
Neo-Cy5 tightly associated with E. coli cells. Levels of Neo-
Cy5 resulting from EDPI-mediated internalization were low
even though it is this fraction that results in the deleterious
effects of aminoglycosides on cells. The loss of membrane
integrity results in the onset of strong diffusive entry of the
drug during EDPII.25,27
The data reported here illustrate how fluorescent derivatives
of an aminoglycoside enable a robust characterization of the
three components of uptake: membrane binding, EDPI, and
EDPII. EDPII-mediated uptake is a consequence of the mis-
coding activity of the drug that accumulated via EDPI. As
miscoding levels can vary between bacterial strains and
between mutants of a single strain, EDPII accumulation may
only partially reflect EDPI accumulation. Thus, it is critical
that the three components of aminoglycoside uptake be eval-
uated. We expect that the method described here will be
applied to characterize aminoglycoside uptake into bacteria
to facilitate a better understanding of the mechanisms of
action of these drugs and their next generation derivatives.
Interestingly, the group of Y. Tor recently investigated the
uptake of several aminoglycoside-Cy3 conjugates including
neomycin-Cy3 and guanidinoneomycin-Cy3 in Chinese
hamster ovarian cells using fluorescence microscopy and
FACS.59 Guanidinoglycosides-Cy3 conjugates entered cells
better than their parent aminoglycosides. This work together
with our results emphasize the usefulness of Cyanine-
aminoglycoside conjugates for further studies of their cellu-
lar uptake by eukaryotic and prokaryotic cells. This class of
antibiotics, including neomycin, has recently drawn strong
attention for the development of novel, potent, and less toxic
drugs to address the pressing societal problem of multidrug-
resistant (MDR) infectious disease.60,61 The approach com-
bining fluorescence microscopy and FACS should provide a
framework for future antibiotic-cell interaction studies and
facilitate the development of novel aminoglycosides.
METHODS
Chemicals. Cy5 and Cy3 NHS-esters were purchased from
General Electric Healthcare or Lumiprobe GmbH. 3,5-
Difluoro-4-hydroxybenzylidene imidazolinone was pur-
chased from Lucerna Technologies. 2,2,2-trifluoro-N-(2-
sulfanylethyl)acetamide was obtained from Merck or pre-
pared following the reported procedure.62
Labeling of neomycin with cyanine fluorophores. A de-
rivative of neomycin containing 2-N-
trifluoroacetamidoethylthio in place of a hydroxyl group in
ring III was used for functionalization. Other amino groups
were protected by hexa-N-tert-butyloxycarbonyl groups for
selective reactivity at the new 5” NH2 position. The deriva-
tive was used to conjugate neomycin to NHS-ester dyes.
Selective deprotection of the 5” position was performed in
ammonium hydroxide in methanol, and the unique free ami-
no group was reacted with Cy5 or Cyanine 5 monofunctional
NHS-ester. Reactions were monitored by analytical thin-
layer chromatography (TLC) on silica gel 60 F254 plates
(0.25 mm). The Cy5 and Cyanine 5 conjugates were purified
on TLC plates (Silica gel 60, Merck), eluted in ethanol, and
deprotected with trifluoroacetic acid (90%) to remove bu-
tyloxycarbonyl groups.
Purification of ribosomes. Tightly coupled 70S ribosomes
from E. coli MRE600 were prepared as described.63,64 Brief-
ly, cells were grown in exponential phase and when the
OD600 reached 0.3, cells were collected by centrifugation.
Cells were lysed by passage through a module/piston ho-
mogenizing system pre-cooled to -80 °C that maintains the
material in a frozen state. The piston was actuated with a
Carver hydraulic press. Ribosomes were isolated by several
centrifugation steps followed by a sucrose gradient (10-40%)
fractionation. The fraction containing 70S particles was
collected using a density gradient fraction system (Teledyne
Isco), pelleted, and resuspended in 50 mM Tris-HCl (pH
7.6), 10 mM MgCl2, 100 mM NH4Cl, 6 mM 2-
mercaptoethanol. Aliquots were stored at -80 °C after flash
freezing in liquid nitrogen. The ribosome concentration was
obtained by measuring the absorption at 260 nm; we as-
sumed that there are 23 pmol ribosomes per A260 unit.
Footprinting of Neo-Cy5 on 70S ribosomes. The concen-
tration of Neo-Cy5 stock solution was estimated from the
absorbance at 649 nm measured in 10 mM Tris-HCl, pH 7.5.
Chemical probing of Neo-Cy5-ribosome complexes was
performed as described.65 We used the RNA base-specific
reagent dimethyl sulfate (DMS) which methylates positions
N1 of adenines and N3 of cytidines as well as position N7 of
guanines.66 Modification reactions (100 µl) with 70S parti-
cles (10 pmol) were performed in a buffer containing 80 mM
potassium cacodylate pH 7.2, 100 mM ammonium chloride,
20 mM magnesium chloride, 1 mM dithiothreitol and 0.5
mM EDTA. In brief, 1 µl of (DMS) (1:10 dilution in 95%
ethanol) was added to a 50-µL reaction mixture. After incu-
bation at 37 °C for 10 minute reactions were stopped by
addition of 25 µL of 6.7% (v/v) β-mercaptoethanol in 280
mM sodium acetate (pH 5.4) and 150 µL of ethanol followed
by rapid mixing. After precipitation, the pellets were resus-
pended in 10 µL of H2O.
Sites of modification were analyzed by fluorescent primer
extension as previously described.67 A Cy5-labeled fluores-
cent DNA primer was purchased from MWG Biotech. Re-
verse transcription reactions were performed in 10 µL of 50
mM Tris-HCl, pH 8.3, 75 mM KCl, 3 mM MgCl2 10 mM
DTT with 0.25 mM each dNTP. SuperScript II (Invitrogen,
0.5 µL 100 units) was added and reactions were incubated at
45 °C for 40 min. For sequencing samples, 1.25 mM of
ddATP, ddCTP, ddGTP, or ddTTP was added to individual
reverse transcription reactions of each sample. Reactions
were stopped by ethanol precipitation. cDNA samples were
dissolved in 10 µL loading solution containing 7 M urea. A
small aliquot (1.5 to 3 µL) of each sample was loaded on an
8% polyacrylamide 7 M urea gel for separation of cDNAs.
Gels were analyzed on a Typhoon imaging system. Molecu-
lar graphics were generated using PyMOL(TM) 2.0.1.
Miscoding assay. Miscoding was assessed in a gain-of-
function assay using firefly and Renilla luciferases. DNA
reporter plasmids pT7 hFluc WT, pT7 hFluc H245R(CGC),
and pT7 hRluc WT for in vitro translation assay were pro-
vided by Erik Böttger and Dimitri Scherbakov (University of
Zurich).5 Wild-type and mutant DNAs were used for in vitro
translation reactions together with the Renilla luciferase
DNA, which served as an internal control. In vitro translation
reactions were performed using the PURExpress system
(New England BioLabs). The premix for one reaction con-
tained 2 µL solution A, 1.5 µL solution B, 5 nM DNA tem-
plates, and 1 unit/µL of SUPERase·In™ RNase Inhibitor
(Invitrogen). The reaction was begun by addition of 4 µL of
the premix lacking aminoglycosides to 1 µL of aminoglyco-
side dilution in 0.2 ml thin-walled tubes on ice. Synthesis
was performed at 37 °C for 35 min in a thermocycler (lid
temperature 98 °C) and stopped by placing samples on ice.
Luciferase activity was measured using the Dual-Luciferase
Reporter Assay System (Promega). Luciferase assay reagent
(50 µl) was added to each sample, and the samples were
mixed and transferred to a white 96-well plate (Greiner,
catalog number 655075) for bioluminescence quantification
using an Infinite M200 microplate reader (Tecan). Mis-
coding was quantified by calculating the mutant fire-
fly/Renilla luciferase activity compared with the wild-type
firefly/Renilla luciferase activity.
Minimum inhibitory concentration measurement. The
minimum inhibitory concentrations (MICs) were determined
using a liquid culture assay according to EUCAST guide-
lines. Aliquots of an overnight culture of E. coli MG1655
were diluted to have an inoculum of 5.5x105 colony-forming
units per mL (c.f.u./mL) into the medium containing 2-fold
serial dilutions of antibiotics. Growth was measured by
turbidity at each concentration during 18 h of incubation at
37 °C. Measurements were performed on 200 µL samples in
a 96-well microtiter plate with transparent lid using an Infi-
nite M200PRO (TECAN). For Neo-Cy5, turbidity was
measured at 400 nm or 500 nm instead of 600 nm to mini-
mize Cy5 excitation.
Treatment of cells with carbonyl cyanide m-chlorophenyl
hydrazone. E. coli MG1655 cells from an overnight culture
in MOPS supplemented with 0.4 % (w/v) glucose (MOPS-G)
were diluted into the same medium. Cells were grown for 2 h
at 37 °C with or without 30 µM carbonyl cyanide m-
chlorophenyl hydrazone (CCCP). The number of live cells at
the time of CCCP addition were estimated as 5x107
c.f.u./mL. After a 4.5-h incubation at 37 °C with neomycin
or Neo-Cy5, cell survival was measured in c.f.u./mL.
Neo-Cy5 uptake. For the liquid culture, MOPS-G was used.
Overnight cultures were diluted to an OD600 of 0.037 with
fresh medium and grown until the OD600 reached 0.2. Typi-
cally, 0.5 µL of Neo-Cy5 dissolved in milliQ water was
added to 5 µL of the cell suspension (diluted 10 times). Cells
were incubated in a heat block at 37 °C without shaking. To
remove Neo-Cy5 not tightly bound or internalized, the sam-
ples were filtered through 0.45 µm-pore-size HAWP mixed
cellulose ester (MCE) membranes (Merck Millipore) mount-
ed on a micro-sample filtration Minifold II (Schleicher &
Schuell) and washed three times with 100 µL MOPS-G pre-
warmed to 37 °C. Immediately after filtration, the section of
the membrane containing cells was placed into an Eppendorf
tube containing MOPS-G, and cells were recovered by gentle
up and down pipetting. Cells were immediately placed on
1% agarose pad for imaging by fluorescence microscopy or
analysis by FACS.
Fluorescence microscopy. Fluorescence images were taken
with an EMCCD camera (Photometrics) through a 63X total
internal reflection fluorescence (TIRF) objective (Zeiss, NA
1.43) mounted on an inverted Zeiss Axio Observer Z1 mi-
croscope. Image acquisition was done with the Metamorph
software package (Molecular Devices). Cy5 illumination was
performed using a 642-nm laser (0.002 kW/cm2, Roper Sci-
entific), and a filter set was used for fluorescence excitation
and emission (Chroma Technology; ET 532/640 nm Laser
Dual Band; excitation filter 530/20 nm and 638/25 nm, di-
chroic mirror 585/70 nm and 650 nm (long pass), and emis-
sion filter 580/70 nm and 700/100 nm). Neo-Cy5 uptake was
typically observed with an electron-multiplying gain of 1 or
50 and an exposure of 10 to 100 ms. Spinach aptamer bound
to 3,5-difluoro-4-hydroxybenzylidene imidazolinone was
excited with a mercury lamp (excitation 470 ± 20 nm, emis-
sion 525 ± 25 nm). Image analyses were performed using
ImageJ and microbeJ.68
FACS analysis. To measure Cy5 fluorescence, cells were
analyzed with a BD Accuri C6 flow cytometer (BD Biosci-
ences). For each experiment, more than 100 000 events were
recorded. Excitation was performed with a 640-nm diode
laser (14.7 mW), and emission was detected through a
675/25 nm band pass filter. Data were analyzed with Kaluza
2.1 (Beckman Coulter) and FlowLogic 7.2. Binding affinity
experiments were analyzed with the software GraphPad
Prism 8.2.1. Data were fitted to a simple saturation curve.
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website.
Supporting methods, 14 figures and one table showing addition-
al results (PDF).
AUTHOR INFORMATION
Corresponding Author
* E-mail: dominique.fourmy@i2bc.paris-saclay.fr.
Present Addresses
† Institute for Environmental and Gender-Specific Medicine,
Juntendo University Graduate School of Medicine, Chiba 279-
0021, Japan.
Author Contributions
S.Y. and D.F. designed the research strategy. M.S.A., M.O. and
D.F performed the microscopy. M.S.A. performed the survival
assays. M.S.A. and D.F performed the FACS and M.R. helped
with analysis. M.O, S.Y. and D.F. performed the biochemistry.
M.C. measured MIC in rich medium on Gram positive and
negative strains and in minimal medium on pathogenic strains.
M.S.A., M.O. and S. Y. performed other MIC experiments. D.F.
performed the chemical synthesis and M.P.H. the NMR and LC-
MS. D.F. wrote the manuscript. All authors have given approval
to the final version of the manuscript.
ACKNOWLEDGMENTS
We wish to thank Marc Mirande, Soufian Ouchane, Anne-Soisig
Steunou, J. Dubois, Stéphanie Bury-Moné for use of equipment.
We wish to thank David Buisson for help with the LC-MS. We
wish to thank Christophe Beloin, Suzana Salcedo, Frédéric
Boccard, Viginia Lioy, Etienne Dervyn, Jean-Luc Pernodet,
Philippe Bouloc for strains and Isabelle Broutin for helpful
discussions. The authors dedicate this paper to the memory of
their colleague Jean-Louis Fourrey. This work was supported by
the Centre National de la Recherche Scientifique (CNRS) to
D.F. and S.Y. M.O. was supported by JASSO, a fellowship from
the French embassy in Japan “Bourse du gouvernement fran-
çais” and a fellowship from FRM (Fondation pour la recherche
Médicale-FDT20140931068). M.S.A. was supported by a PhD
fellowship from the French Ministère de l’Enseignement Supé-
rieur et de la Recherche (MESR).
REFERENCES
(1) Jackson, J.; Chen, C.; Buising, K. Aminoglycosides: How
Should We Use Them in the 21st Century? Curr. Opin. Infect.
Dis. 2013, 26 (6), 516–525.
(2) Takahashi, Y.; Igarashi, M. Destination of Aminoglycoside
Antibiotics in the “Post-Antibiotic Era.” J. Antibiot. 2018, 71,
4–14.
(3) Aggen, J. B.; Armstrong, E. S.; Goldblum, A. A.; Dozzo, P.;
Linsell, M. S.; Gliedt, M. J.; Hildebrandt, D. J.; Feeney, L. A.;
Kubo, A.; Matias, R. D.; Lopez, S.; Gomez, M.; Wlasichuk, K.
B.; Diokno, R.; Miller, G. H.; Moser, H. E. Synthesis and Spec-
trum of the Neoglycoside ACHN-490. Antimicrob. Agents
Chemother. 2010, 54 (11), 4636–4642.
(4) Huth, M. E.; Han, K.-H.; Sotoudeh, K.; Hsieh, Y.-J.; Effertz,
T.; Vu, A. A.; Verhoeven, S.; Hsieh, M. H.; Greenhouse, R.;
Cheng, A. G.; Ricci, A. J. Designer Aminoglycosides Prevent
Cochlear Hair Cell Loss and Hearing Loss. J. Clin. Invest. 2015,
125 (2), 583–592.
(5) Matt, T.; Ng, C. L.; Lang, K.; Sha, S.-H.; Akbergenov, R.;
Shcherbakov, D.; Meyer, M.; Duscha, S.; Xie, J.; Dubbaka, S.
R.; Perez-Fernandez, D.; Vasella, A.; Ramakrishnan, V.;
Schacht, J.; Böttger, E. C. Dissociation of Antibacterial Activity
and Aminoglycoside Ototoxicity in the 4-Monosubstituted 2-
Deoxystreptamine Apramycin. Proc. Natl. Acad. Sci. U.S.A.
2012, 109 (27), 10984–10989.
(6) Sato, R.; Tanigawara, Y.; Kaku, M.; Aikawa, N.; Shimizu,
K. Pharmacokinetic-Pharmacodynamic Relationship of Arbeka-
cin for Treatment of Patients Infected with Methicillin-Resistant
Staphylococcus Aureus. Antimicrob. Agents Chemother. 2006,
50 (11), 3763–3769.
(7) Zhanel, G. G.; Lawson, C. D.; Zelenitsky, S.; Findlay, B.;
Schweizer, F.; Adam, H.; Walkty, A.; Rubinstein, E.; Gin, A. S.;
Hoban, D. J.; Lynch, J. P.; Karlowsky, J. A. Comparison of the
Next-Generation Aminoglycoside Plazomicin to Gentamicin,
Tobramycin and Amikacin. Expert Rev Anti Infect Ther 2012,
10 (4), 459–473.
(8) Borovinskaya, M. A.; Pai, R. D.; Zhang, W.; Schuwirth, B.
S.; Holton, J. M.; Hirokawa, G.; Kaji, H.; Kaji, A.; Cate, J. H.
Structural Basis for Aminoglycoside Inhibition of Bacterial
Ribosome Recycling. Nat Struct Mol Biol 2007, 14 (8), 727–
732.
(9) Campuzano, S.; Vazquez, D.; Modolell, J. Functional Inter-
action of Neomycin B and Related Antibiotics with 30S and 50S
Ribosomal Subunits. Biochem Biophys Res Commun 1979, 87
(3), 960–966.
(10) Moazed, D.; Noller, H. F. Interaction of Antibiotics with
Functional Sites in 16S Ribosomal RNA. Nature 1987, 327
(6121), 389–394.
(11) Davies, J.; Gorini, L.; Davis, B. D. Misreading of RNA
Codewords Induced by Aminoglycoside Antibiotics. Mol Phar-
macol 1965, 1 (1), 93–106.
(12) Carter, A. P.; Clemons, W. M.; Brodersen, D. E.; Morgan-
Warren, R. J.; Wimberly, B. T.; Ramakrishnan, V. Functional
Insights from the Structure of the 30S Ribosomal Subunit and
Its Interactions with Antibiotics. Nature 2000, 407 (6802), 340–
348.
(13) Demeshkina, N.; Jenner, L.; Westhof, E.; Yusupov, M.;
Yusupova, G. A New Understanding of the Decoding Principle
on the Ribosome. Nature 2012, 484 (7393), 256–259.
(14) Fourmy, D.; Recht, M. I.; Blanchard, S. C.; Puglisi, J. D.
Structure of the A Site of Escherichia Coli 16S Ribosomal RNA
Complexed with an Aminoglycoside Antibiotic. Science 1996,
274 (5291), 1367–1371.
(15) Vicens, Q.; Westhof, E. Crystal Structure of Paromomycin
Docked into the Eubacterial Ribosomal Decoding A Site. Struc-
ture 2001, 9 (8), 647–658.
(16) Yoshizawa, S.; Fourmy, D.; Puglisi, J. D. Structural Origins
of Gentamicin Antibiotic Action. Embo J 1998, 17 (22), 6437–
6448.
(17) Cabanas, M. J.; Vazquez, D.; Modolell, J. Inhibition of
Ribosomal Translocation by Aminoglycoside Antibiotics. Bio-
chem Biophys Res Commun 1978, 83 (3), 991–997.
(18) Misumi, M.; Nishimura, T.; Komai, T.; Tanaka, N. Interac-
tion of Kanamycin and Related Antibiotics with the Large Sub-
unit of Ribosomes and the Inhibition of Translocation. Biochem
Biophys Res Commun 1978, 84 (2), 358–365.
(19) Hirokawa, G.; Kiel, M. C.; Muto, A.; Selmer, M.; Raj, V.
S.; Liljas, A.; Igarashi, K.; Kaji, H.; Kaji, A. Post-Termination
Complex Disassembly by Ribosome Recycling Factor, a Func-
tional TRNA Mimic. Embo J 2002, 21 (9), 2272–2281.
(20) Hirokawa, G.; Kaji, H.; Kaji, A. Inhibition of Antiassocia-
tion Activity of Translation Initiation Factor 3 by Paromomycin.
Antimicrob Agents Chemother 2007, 51 (1), 175–180.
(21) Wallace, B. J.; Tai, P. C.; Herzog, E. L.; Davis, B. D. Par-
tial Inhibition of Polysomal Ribosomes of Escherichia Coli by
Streptomycin. Proc Natl Acad Sci U S A 1973, 70 (4), 1234–
1237.
(22) Foster, C.; Champney, W. S. Characterization of a 30S
Ribosomal Subunit Assembly Intermediate Found in Escherich-
ia Coli Cells Growing with Neomycin or Paromomycin. Arch
Microbiol 2008, 189 (5), 441–449.
(23) Mehta, R.; Champney, W. S. 30S Ribosomal Subunit As-
sembly Is a Target for Inhibition by Aminoglycosides in Esche-
richia Coli. Antimicrob Agents Chemother 2002, 46 (5), 1546–
1549.
(24) Sykes, M. T.; Shajani, Z.; Sperling, E.; Beck, A. H.; Wil-
liamson, J. R. Quantitative Proteomic Analysis of Ribosome
Assembly and Turnover in Vivo. J Mol Biol 2010, 403 (3), 331–
345.
(25) Davis, B. D. Mechanism of Bactericidal Action of Amino-
glycosides. Microbiol Rev 1987, 51 (3), 341–350.
(26) Hancock, R. E. Aminoglycoside Uptake and Mode of
Action-with Special Reference to Streptomycin and Gentamicin.
II. Effects of Aminoglycosides on Cells. J. Antimicrob.
Chemother. 1981, 8 (6), 429–445.
(27) Taber, H. W.; Mueller, J. P.; Miller, P. F.; Arrow, A. S.
Bacterial Uptake of Aminoglycoside Antibiotics. Microbiol Rev
1987, 51 (4), 439–457.
(28) Bryan, L. E.; Kwan, S. Mechanisms of Aminoglycoside
Resistance of Anaerobic Bacteria and Facultative Bacteria
Grown Anaerobically. J. Antimicrob. Chemother. 1981, 8 Suppl
D, 1–8.
(29) Damper, P. D.; Epstein, W. Role of the Membrane Poten-
tial in Bacterial Resistance to Aminoglycoside Antibiotics.
Antimicrob. Agents Chemother. 1981, 20 (6), 803–808.
(30) Eisenberg, E. S.; Mandel, L. J.; Kaback, H. R.; Miller, M.
H. Quantitative Association between Electrical Potential across
the Cytoplasmic Membrane and Early Gentamicin Uptake and
Killing in Staphylococcus Aureus. J. Bacteriol. 1984, 157 (3),
863–867.
(31) Davis, B. D.; Chen, L. L.; Tai, P. C. Misread Protein Cre-
ates Membrane Channels: An Essential Step in the Bactericidal
Action of Aminoglycosides. Proc Natl Acad Sci U S A 1986, 83
(16), 6164–6168.
(32) Hancock, R. E. Aminoglycoside Uptake and Mode of
Action with Special Reference to Streptomycin and Gentamicin.
I. Antagonists and Mutants. J. Antimicrob. Chemother. 1981, 8
(4), 249–276.
(33) Peng, B.; Su, Y.-B.; Li, H.; Han, Y.; Guo, C.; Tian, Y.-M.;
Peng, X.-X. Exogenous Alanine and/or Glucose plus Kanamy-
cin Kills Antibiotic-Resistant Bacteria. Cell Metab. 2015, 21 (2),
249–262.
(34) Stone, M. R. L.; Butler, M. S.; Phetsang, W.; Cooper, M.
A.; Blaskovich, M. A. T. Fluorescent Antibiotics: New Research
Tools to Fight Antibiotic Resistance. Trends Biotechnol. 2018,
36 (5), 523–536.
(35) Allison, K. R.; Brynildsen, M. P.; Collins, J. J. Metabolite-
Enabled Eradication of Bacterial Persisters by Aminoglycosides.
Nature 2011, 473 (7346), 216–220.
(36) Cinquin, B.; Maigre, L.; Pinet, E.; Chevalier, J.; Stavenger,
R. A.; Mills, S.; Réfrégiers, M.; Pagès, J.-M. Microspectromet-
ric Insights on the Uptake of Antibiotics at the Single Bacterial
Cell Level. Sci Rep 2015, 5, 17968.
(37) Vergalli, J.; Dumont, E.; Pajović, J.; Cinquin, B.; Maigre,
L.; Masi, M.; Réfrégiers, M.; Pagés, J.-M. Spectrofluorimetric
Quantification of Antibiotic Drug Concentration in Bacterial
Cells for the Characterization of Translocation across Bacterial
Membranes. Nat Protoc 2018, 13 (6), 1348–1361.
(38) Dumont, E.; Vergalli, J.; Conraux, L.; Taillier, C.; Vassort,
A.; Pajovic, J.; Réfrégiers, M.; Mourez, M.; Pagès, J.-M. Anti-
biotics and Efflux: Combined Spectrofluorimetry and Mass
Spectrometry to Evaluate the Involvement of Concentration and
Efflux Activity in Antibiotic Intracellular Accumulation. J.
Antimicrob. Chemother. 2019, 74 (1), 58–65.
(39) Hernandez Linares, A.; Fourmy, D.; Fourrey, J.-L.;
Loukaci, A. Introduction of a Substituent at the 5"-Position of
N-Boc Neomycin B under Mitsunobu Reaction Conditions. Org.
Biomol. Chem. 2005, 3 (11), 2064–2066.
(40) Dempsey, G. T.; Vaughan, J. C.; Chen, K. H.; Bates, M.;
Zhuang, X. Evaluation of Fluorophores for Optimal Perfor-
mance in Localization-Based Super-Resolution Imaging. Nat
Methods 2011, 8 (12), 1027–1036.
(41) Recht, M. I.; Douthwaite, S.; Puglisi, J. D. Basis for Pro-
karyotic Specificity of Action of Aminoglycoside Antibiotics.
Embo J 1999, 18 (11), 3133–3138.
(42) Pfister, P.; Hobbie, S.; Brull, C.; Corti, N.; Vasella, A.;
Westhof, E.; Bottger, E. C. Mutagenesis of 16S RRNA C1409-
G1491 Base-Pair Differentiates between 6’OH and 6’NH3+
Aminoglycosides. J Mol Biol 2005, 346 (2), 467–475.
(43) Perez-Fernandez, D.; Shcherbakov, D.; Matt, T.; Leong, N.
C.; Kudyba, I.; Duscha, S.; Boukari, H.; Patak, R.; Dubbaka, S.
R.; Lang, K.; Meyer, M.; Akbergenov, R.; Freihofer, P.; Vaddi,
S.; Thommes, P.; Ramakrishnan, V.; Vasella, A.; Böttger, E. C.
4’-O-Substitutions Determine Selectivity of Aminoglycoside
Antibiotics. Nat Commun 2014, 5, 3112.
(44) Perzynski, S.; Cannon, M.; Cundliffe, E.; Chahwala, S. B.;
Davies, J. Effects of Apramycin, a Novel Aminoglycoside Anti-
biotic on Bacterial Protein Synthesis. Eur. J. Biochem. 1979, 99
(3), 623–628.
(45) Hancock, R. E.; Farmer, S. W.; Li, Z. S.; Poole, K. Interac-
tion of Aminoglycosides with the Outer Membranes and Puri-
fied Lipopolysaccharide and OmpF Porin of Escherichia Coli.
Antimicrob Agents Chemother 1991, 35 (7), 1309–1314.
(46) Okuda, M.; Fourmy, D.; Yoshizawa, S. Use of Baby Spin-
ach and Broccoli for Imaging of Structured Cellular RNAs.
Nucleic Acids Res. 2017, 45 (3), 1404–1415.
(47) Jörgensen, F.; Kurland, C. G. Death Rates of Bacterial
Mutants. FEMS Microbiology Letters 1987, 40 (1), 43–46.
(48) White, J. R.; White, H. L. Streptomycinoid Antibiotics:
Synergism by Puromycin. Science 1964, 146 (3645), 772–774.
(49) Klainer, A. S.; Russell, R. R. Effect of the Inhibition of
Protein Synthesis on the Escherichia Coli Cell Envelope. Anti-
microb. Agents Chemother. 1974, 6 (2), 216–224.
(50) Ezraty, B.; Vergnes, A.; Banzhaf, M.; Duverger, Y.; Hu-
guenot, A.; Brochado, A. R.; Su, S. Y.; Espinosa, L.; Loiseau,
L.; Py, B.; Typas, A.; Barras, F. Fe-S Cluster Biosynthesis
Controls Uptake of Aminoglycosides in a ROS-Less Death
Pathway. Science 2013, 340 (6140), 1583–1587.
(51) Bryan, L. E.; Elzen, H. M. V. D. Streptomycin Accumula-
tion in Susceptible and Resistant Strains of Escherichia Coli and
Pseudomonas Aeruginosa. Antimicrob. Agents Chemother.
1976, 9 (6), 928–938.
(52) Aires, J. R.; Köhler, T.; Nikaido, H.; Plésiat, P. Involve-
ment of an Active Efflux System in the Natural Resistance of
Pseudomonas Aeruginosa to Aminoglycosides. Antimicrob.
Agents Chemother. 1999, 43 (11), 2624–2628.
(53) Sobel, M. L.; McKay, G. A.; Poole, K. Contribution of the
MexXY Multidrug Transporter to Aminoglycoside Resistance in
Pseudomonas Aeruginosa Clinical Isolates. Antimicrob. Agents
Chemother. 2003, 47 (10), 3202–3207.
(54) Westbrock-Wadman, S.; Sherman, D. R.; Hickey, M. J.;
Coulter, S. N.; Zhu, Y. Q.; Warrener, P.; Nguyen, L. Y.;
Shawar, R. M.; Folger, K. R.; Stover, C. K. Characterization of
a Pseudomonas Aeruginosa Efflux Pump Contributing to Ami-
noglycoside Impermeability. Antimicrob. Agents Chemother.
1999, 43 (12), 2975–2983.
(55) Li, X.-Z.; Nikaido, H. Efflux-Mediated Drug Resistance in
Bacteria. Drugs 2004, 64 (2), 159–204.
(56) Monlezun, L.; Phan, G.; Benabdelhak, H.; Lascombe, M.-
B.; Enguéné, V. Y. N.; Picard, M.; Broutin, I. New OprM Struc-
ture Highlighting the Nature of the N-Terminal Anchor. Front
Microbiol 2015, 6, 667.
(57) Nakashima, R.; Sakurai, K.; Yamasaki, S.; Nishino, K.;
Yamaguchi, A. Structures of the Multidrug Exporter AcrB
Reveal a Proximal Multisite Drug-Binding Pocket. Nature 2011,
480 (7378), 565–569.
(58) Murakami, S.; Nakashima, R.; Yamashita, E.; Matsumoto,
T.; Yamaguchi, A. Crystal Structures of a Multidrug Transporter
Reveal a Functionally Rotating Mechanism. Nature 2006, 443
(7108), 173–179.
(59) Hadidi, K.; Wexselblatt, E.; Esko, J. D.; Tor, Y. Cellular
Uptake of Modified Aminoglycosides. J. Antibiot. 2018, 71,
142–145.
(60) Böttger, E. C.; Crich, D. Aminoglycosides: Time for the
Resurrection of a Neglected Class of Antibacterials? ACS Infect
Dis 2020, 6 (2), 168-172.
(61) Sati, G. C.; Sarpe, V. A.; Furukawa, T.; Mondal, S.; Man-
tovani, M.; Hobbie, S. N.; Vasella, A.; Böttger, E. C.; Crich, D.
Modification at the 2’-Position of the 4,5-Series of 2-
Deoxystreptamine Aminoglycoside Antibiotics To Resist Ami-
noglycoside Modifying Enzymes and Increase Ribosomal Tar-
get Selectivity. ACS Infect Dis 2019, 5 (10), 1718–1730.
(62) Jagt, R. B. C.; Gómez-Biagi, R. F.; Nitz, M. Pattern-Based
Recognition of Heparin Contaminants by an Array of Self-
Assembling Fluorescent Receptors. Angew. Chem. Int. Ed. Engl.
2009, 48 (11), 1995–1997.
(63) Mazauric, M. H.; Leroy, J. L.; Visscher, K.; Yoshizawa, S.;
Fourmy, D. Footprinting Analysis of BWYV Pseudoknot-
Ribosome Complexes. Rna 2009, 15 (9), 1775–1786.
(64) Powers, T.; Noller, H. F. A Functional Pseudoknot in 16S
Ribosomal RNA. Embo J 1991, 10 (8), 2203–2214.
(65) Yoshizawa, S.; Fourmy, D.; Puglisi, J. D. Recognition of
the Codon-Anticodon Helix by Ribosomal RNA. Science 1999,
285 (5434), 1722–1725.
(66) Fourmy, D.; Yoshizawa, S. Protein-RNA Footprinting: An
Evolving Tool. Wiley Interdiscip Rev RNA 2012, 3 (4), 557–
566.
(67) Ying, B. W.; Fourmy, D.; Yoshizawa, S. Substitution of the
Use of Radioactivity by Fluorescence for Biochemical Studies
of RNA. Rna 2007, 13 (11), 2042–2050.
(68) Ducret, A.; Quardokus, E. M.; Brun, Y. V. MicrobeJ, a
Tool for High Throughput Bacterial Cell Detection and Quanti-
tative Analysis. Nat Microbiol 2016, 1 (7), 16077.
 For Table of Contents Only
Fluorescent aminoglycoside antibiotics and methods for accurately monitoring uptake by bacteria

Mahnaz Sabeti Azad, Maho Okuda, Mélina Cyrenne, Mickael Bourge, Marie-Pierre Heck, Satoko Yo-
shizawa, and Dominique Fourmy.

brief synopsis, describing the graphic and explaining how the manuscript relates to sustainability:

Fluorescently labeled aminoglycoside derivatives with uptake and bactericidal activities similar to those
of the parent drug were generated for facilitating studies involving aminoglycoside antibiotics. Robust
characterization
Structures corrigées :of the drug uptake is enabled by the method which combines fluorescence microscopy
with fluorescence-activated
Structures corrigées :
cell sorting (FACS) using neomycin coupled to non-permeable cyanine
dyes.
Neomycin
Neomycin

FACS Background
Cell OD600 0.3
Cell OD600 0.03
Neo-Cy5Neo-Cy5 Cell OD600 0.003

Count
Intermediate
Intermediate for
Cy5
NHS Cy5
for coupling NHS Cy5
coupling P % C %
0,3 93,39 6,61
0,03 55,12 44,88
0,003 32,15 66,22
Figure S3. Scheme for generation of Neo-Cy5. The primary amine group that reacts
Neo-Cy5
with the NHS-ester-Cy5 is highlighted by the red circle. Other amino groups are
Aminoglycoside antibiotic Figure 8
Figure S3. protected by hexa-N-tert-butyloxycarbonyl groups.
Scheme for generation of Neo-Cy5. The primary amine group that reacts Neo-Cy5 Neo-Cy3 Okuda et al., 2018
with the NHS-ester-Cy5 is highlighted by the red circle. Other amino groups are
protected by hexa-N-tert-butyloxycarbonyl groups.
Supporting Information

Fluorescent aminoglycoside antibiotics and methods for accurately monitoring
uptake by bacteria

Mahnaz Sabeti Azad†, Maho Okuda†,‡, Mélina Cyrenne†, Mickael Bourge†, Marie-Pierre
Heck§, Satoko Yoshizawa†, and Dominique Fourmy*,†.
† Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC),

91198, Gif-sur-Yvette, France.

§ Université Paris-Saclay, CEA, Service de Chimie Bio-organique et de Marquage, 91191

Gif-sur-Yvette, France.
Corresponding author
*E-mail : dominique.fourmy@i2bc.paris-saclay.fr

17 pages
Supporting methods
14 figures
1 tables:

S1

Supporting methods
Products were verified by 1H and 13C Nuclear Magnetic Resonance spectroscopy (NMR)
or Liquid Chromatography Mass Spectrometry (LC-MS). NMR could not be performed for
Neo-Cy5 as the quantities synthetized were too low.

Synthetic Procedures and characterization data

1,3,2',6',2''',6''' Hexa-N-tert-butyloxycarbonylneomycin 2: To a solution of
commercially available Neomycin sulfate (1 g, 1,4 mmol) in DMF/H2O (20 mL/20 mL)
were added triethylamine (9 mL) and di-tert-butyl dicarbonate (2,1 g, 9,62 mmol, 6.9
eq.). The mixture was heated at 60° C for 5 h. After concentration under vacuum, the
crude was dissolved in EtOAc and washed with brine. The organic layer was recovered
and dried on MgSO4. After concentration and purification of the crude by silicagel
column chromatography (CH2Cl2/MeOH (9/1)) 2 was isolated (876 mg, 52 % yield; oil).
1H NMR (300 MHz, CD OD) d = 5.45 (br, 1H, H1'), 5.25 (br, 1H, H1''), 5.10 (br, 1H, H1'''),
3
4.9-4.8 (m, 2H), 4.2-4.11 (br, 2H), 3.9 (m, 1H), 3.85-3.78 (m, 3H), 3.76-3.55 (m, 5H), 3.52-
3.39 (m, 6H), 3.32-3.22 (m, 5H), 1.90 (br, 1H, Heq), 1.49-1.35 (m, 54H), 1.3 (br, 1H,
Hax);13C NMR (75 MHz, CD3OD) d = 158.8, 158.3, 158.0, 157.8; 110.5, 100.2, 99.2, 87.6,
83.3, 80.6, 78.2, 75.6, 74.3, 72.9, 72.4, 71.5, 69.0, 63.4, 57.1, 53.4, 52.2, 51.7, 42.6, 41.7,
35.8, 28.9. MS (ESI+): m/z calculated for [C53H94N6O25+Na]+: 1237,6, found : 1237,4;
elemental Analysis calculated C, 52.38; H, 7.80; N, 6.92; O, 32.91; found C, 49.95; H, 7.91;
N, 6.55; O, 30.86.

5"-O-triisopropylbenzensulfonyl-1,3,2',6',2"',6"'-hexa-N-tert-butyloxycarbonyl
Neomycin 3: To a solution of compound 2 (500 mg, 0.41 mmol) in pyridine (3 mL) was
added a solution of 2,4,6 triisopropylbenzenesulfonyl chloride (4 g) in pyridine (7 mL).
The solution was stirred at RT for 48 h and was concentrated under vaccuum. The crude
was dissolved in EtOAc and washed with brine. The organic layer was recovered, dried
on MgSO4. After concentration and purification by silicagel column chromatography
(CH2Cl2/MeOH (10/1)) 3 was isolated (432 mg, 71 % yield); 1H NMR (300 MHz, CD3OD)
d = 7.01 (s, 2H, Har), 6.1 (br, 1H, H1'), 5.18 (br, 1H, Heq), 4.9 (br, 1H, H1"'), 4.65-3.79 (m,

S2

8H), 4.3-4.0(m, 2H), 3.8-3.6 (m, 8H), 3.4-3.2 (m, 4H), 2.7 (br, 1H, Heq), 2.92-2.85 (m, 3H),
1.69 (br, 1H, Hax), 1.9-1.3 (m, 55H) . 1.18 (s, 18H); 13C NMR (75 MHz, CD3OD) d = 169.5,
159.4, 158.7, 158.5, 158.2, 157.7, 132.8, 132.4, 130.9, 130.2, 125.5, 111.1, 100.0, 98.6,
87.0, 81.1, 80.8, 80.7, 80.3, 79.2, 75.8, 74.8, 73.2, 72.8, 71.8, 71.2, 69.8, 69.4, 68.8, 68.5,
56.7, 53.7, 52.6, 42.7, 41.6, 35.7, 29.3, 28.9, 25.7, 24.2, 25.0; MS (ESI+): m/z calculated for
[C68H116N6O27S1+Na+H]+ : 1504,7; found : 1504.8, [C68H116N6O27S1+2Na]2+ : 763.35,
found : 763.5.

5"-(2-N-trifluoroacetamidoethylthiol)-1,3,2',6',2"',6"'-Hexa-N-tert-
butyloxycarbonyl-5"'-deoxyneomycin 4: To a solution of sodium ethoxide (4 mL, 3 M
in EtOH) under Argon was added commercially available 2,2,2-trifluoro-N-(2-
mercaptoethyl)acetamide (90 mg, 0.51 mmol) in EtOH (3 mL) and the mixture was
stirred at RT for 15 min. Then was added a solution of compound 3 (220 mg, 0.15 mmol)
in EtOH (3 mL) and the mixture was stirred at RT for 5 h. 20 mL of CH2Cl2 were added,
and then an aqueous solution of sodium phosphate (pH = 6). The organic layer was
separated, dried on MgSO4 and concentrated. The crude was purified by silicagel column
chromatography (CH2Cl2/MeOH (10/1)) to afford 4 (184 mg, 91 % yield); 1H NMR (300
MHz, CD3OD) d = 6.75 (m, 1H), 6.52 (m, 2H), 6.15 (br, 1H, H1'), 5.45 (br, 1H, H1"), 5.21
(br, 1H, H1"'), 4.8-4.3 (m, 6H), 4.28-4.15 (m, 2H), 4.08-4.0 (m, 1H), 3.8-3.75 (m, 2H),
3.72-3.63 (m, 2H), 3.59-3.45 (m, 6H), 3.39-3.22 (m, 4H), 2.81-2.79(m, 2H), 2.76-2.70 (m,
2H), 2.03-1.96 (m, 1H, Heq), 1.78-1.74(m, 1H), 1.52-1.48 (m, 54H+Hax); MS (ESI-TOF):
m/z calculated for [C57H98F3N7O25S1+1H]+ :1371,5 found : 1371,5
[[C57H98F3N7O25S1+1H] : 686.3, found : 686.4.
2+

5"-(2-aminoethylthio-)-1,3,2',6',2"',6"'-Hexa-N-tert-butyloxycarbonyl-5"-
deoxyneomycin 5:
Compound 4 (84 mg, 0.061 mmol) was dissolved in NH4OH solution in MeOH (3 mL,
70%). The solution was strirred at TA for 1 h and then concentrated under vaccum to

S3

afford 5 (36 mg, 46% yield). 1H NMR (300 MHz, CD3OD) d =5.44 (br, 1H, H1'), 5.2 (br,
1H, H1"'), 4.92 (br, 1H, H1"'), 1H), 4.34 (m, 1H), 4.22(m, 1H), 4.05 (m, 1H), 3.9 (m, 2H),
3.75(m, 2H), 3.64-3.12 (m, 15H), 3.0-2.78 (m, 4H), 1.95 (br, 1H, Heq), 1.68 (m, 1H, Hax),
1.64-1.38 (m, 54H); MS (ESI-TOF): m/z calculated for [C55H99N7O24S1+2H]+ :1276,5
found : 1276,06, [[C55H99N7O24S1+2H:]2+ : 638.5 found : 638.45.

Neo-Cy5


The compound 5 was resuspended in 100 µL of dimethyl formamide (DMF). N,N-
Diisopropylethylamine (100 µL) was added to two micromoles of Cy5 (GE Healthcare)
or Cyanine 5 (Lumiprobe) monofunctional NHS-ester previously dissolved in 500 µL
DMF. The sample was next placed for two hours at room temperature with the Boc-
neomycin derivative for coupling. The reaction was monitored by analytical thin-layer
chromatography (TLC) on silica gel 60 F254 plates with eluant:
chloroform/Ethanol/NH4OH 5.6/3.8/0.45 (Figure S6c). Then the sample was dried
under vacuum and kept at – 80°C if necessary. The product was dissolved in methanol
for purification on TLC plates (Silica gel 60, Merck). The silica gel was recovered and
placed in Eppendorf tubes and the product eluted in ethanol. The resin and the solvent
were separated by centrifugation and the elution step repeated 3 times. The sample was
dried and finally, removal of the butyloxycarbonyl groups was performed in
trifluoroacetic acid (90%) for 10 minutes at room temperature.

S4

 R21 =

11a, 11b OH
O
H2N
R3
O HN
12a, 12b (R3= H)
HO
HO H2N O N
Paromomycin
H2N
O NH2
O 13a, 13b (R3= CH3 ) O
R1 O
0.01 +M

OH
0.1 +M
0.5 +M

2.5 +M
- DMS

10 +M

Me
0 +M

1 +M

5 +M

HN
UGCA
R2 OH 14a, 14b
O N
NH

a : R 2 = OH G1494 O
O
H2N O
NH2 Me
b : R2 =
HN
HO 15a, 15b
HO O
Manip du 9/03/07 H O N
H
N
Neomycin Neomycin U Paromomycin after Chloro S
extraction O
0.01 +M

0.1 +M
0.5 +M
0.1 +M
0.5 +M

2.5 +M
0.1 +M
0.5 +M

2.5 +M

16b NH2
10 +M
20 +M
50 +M
- DMS

10 +M

1 +M

5 +M
0 +M

1 +M

5 +M

Figure S1. Derivatives of neomycin. UGCA

Sup Figure 7
Table S1 : MIC values for neomycin derivatives against E. coli MG1655 cells grown
in LB medium.
product N° R1 MIC μg/ml
11b OH 5
G1494
12b uracil 10
13b thymine >40
14b thyminylacetyl 20
15b thyminylacetamidoethylthio 5
16b NH2 >40

Gel du 20/03/07

Neomycin Neo-U
0.01 +M

0.5 +M

2.5 +M
0.5 +M

2.5 +M

10 +M
- DMS

10 +M
0 +M

1 +M

5 +M
0 +M

1 +M

5 +M

A1492
G1494

S5


Figure S2. DMS probing of 30S ribosomal subunits bound to neomycin and Neo-U. The
lane marked “- DMS” is a control reaction with no DMS added. Bands corresponding to
Structures corrigées :
nucleotides A1492 and G1494 are indicated.

Neomycin

Neo-Cy5

Intermediate
for NHS Cy5
coupling

Figure S3. Scheme for generation of Neo-Cy5. The primary amine group that reacts
Figure S3. Scheme for generation of Neo-Cy5. The primary amine group that reacts with
with the NHS-ester-Cy5 is highlighted by the red circle. Other amino groups are
the NHS-ester-Cy5 is highlighted by the red circle. Other amino groups are protected by
protected by hexa-N-tert-butyloxycarbonyl groups.
N-tert-butyloxycarbonyl groups.

52 % yield 71 % yield

Neomycin 1

46 % yield
91 % yield

Conditions and reagents :

a) TEA, Boc2O, DMF, 60°C; NH4OH, MeOH; b) TIPBSCl, Py, RT 48 h; c) EtONa, trifluoro-N-(2-mercaptoethyl)acetamide, RT, 5 h; d) NH4OH, MeOH


Figure S4. Synthesis of Boc-Neo-Cy5 from Boc-Neomycin. All steps were performed at
LE 1e rendement
room temperature.
est a 52 % après
calcul

S6

 a) b)
rrigées :

6 Neo-Cy5

Conditions and reagents : a) NH4OH, MeOH, b) i) NHS-Cy5, DIEA, DMF; ii) TFA/H2O 9/1

Neo-Cy5Neo-Cy5

I noted a mistake on feb 10, the midle structure is haveving Cy5 GE and final one Cy5 lumiprobe. I put a white box to correct
NHS Cy5
NHS Cy5

Neo-Cy5 (GE Healthcare) Neo-Cy5 (Lumiprobe)

eneration of Neo-Cy5. TheS5.
Figure primary amine group
Synthesis that reacts
of Boc-Neo-Cy5 from Boc-Neomycin. All steps were performed at
is highlighted by the red circle. Other amino groups are
butyloxycarbonyl
room temperature. Below are indicated the two structures of Neo-Cy5 according to the
n of Neo-Cy5.groups.
The primary amine group that reacts
source of Cy5-NHS (GE Healthcare or Lumiprobe). The difference is highlighted by an
lighted by the red circle. Other amino groups are
arrow.

ycarbonyl groups.

S7

 Néomycine DF 63

a)

Chemical Formula: C57H98F3N7O25S

MS(ESI-TOF) m/z calcd for [C57H98F3N7O25S +1H]+ = 1371,5 found = 1371,5

Sup Figur
et j'ai aussi son 1H RMN

S8

 b) MS obtained using an ESI-Quadripole autopurify (pump : 2545, mass: ZQ2000) mass spectrometer.

MS(ESI-TOF) m/z calcd for [C55H99N7O24S1+2H]+ = 1276,5, found = 1276,06

MH450b2 411 (3.011) Cm (405:432) 1: Scan ES+

100
1276.06 1.91e7
638.45 19132344
17661404
%

588.37
8173135

1275.30
560.31 4674288
4008259

124.07 532.29 667.93

1345984 1400102 1443313
214.18
869.00 914.77 988.67 1273.35 1278.52 1914.23 1953.63
328363 503.92;324765 668.94;85413 1163.41 18765 276617 1394.96 1559.25 1701.16 1839.64 477468 4927
89978 17156 5095 2735 6552 6118 62490 4822
0 m/z
200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900
MH450b2 (0.003) Is (1.00,1.00) C55H99N7O24S 1: Scan ES+
100
116.79 319.42 637.83 1274.65 4.78e12
4778909237248 4778909237248 4778909237248 4778909237248

425.89 638.33 1275.66

3100853665792 3100853665792 3100853665792
%

426.22 638.83 1276.66

1439767068672 1439767068672 1439767068672

426.56 639.33 1277.66

497368662016 497368662016 497368662016

426.89;140700057600 639.83;140700057600 1278.66;140700057600

0 m/z
200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900
MH450b2 411 (3.011) Cm (409:420) 1: Scan ES+
100
1276.09 2.82e7
28204382

638.43
20496154
%

588.41
9886631 1275.32
7899083

560.29
4904662

532.33
1724265 667.80
119.99 214.25 1018006 1032.99 1273.29 1278.50 1447.42 1738.90 1914.28 1958.06
850.85 914.77 981.39 1700.18
407787 200057 460.14;108542 878 1152.63 25037 351592 1394.96 12527 1561.12 4804 900470 5128
73349 19188 4078 566 14634 5949 74418
0 m/z

200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900

S9

 c) Loading Neo-Cy5 Neo-Cy3

Neo-Cy5
Cy5-NHS

Neo-Cy3

Cy3-NHS

d) Neo-Cy5

Neo-Cy5
Produit final Neo Cy 5


e)
Sup Figure xx

Neo-Cy5

MS obtained using an ESI-Quadripole autopurify (pump : 2545, mass: ZQ2000) mass spectrometer.

MS(ESI-TOF) m/z calcd for [C57H87N9O19S3-1H]- = 1297,54, found = 1297,56; [C57H87N9O19S3-2H]2-=

648.75, found = 648,37; [C57H87N9O19S3+3H]3+ = 433,67, found = 434,01.


NEO-CY5-S4 513 (3.759) Cm (477:640) 1: Scan ES+

100
434.01 8.67e6

S10

%

Sup
 MS(ESI-TOF) m/z calcd for [C57H87N9O19
648.75, found = 648,37; [C57H87N9O19S3

NEO-CY5-S4 513 (3.759) Cm (477:640)
434.01
100
%

214.27

124.18

336.14
650.29

230.79

346.32
438.72 505.10
642.49 651.19
231.61 325.85
130.62
158.26 389.58 460.55 505.92 652.01
251.28 325.13 603.72 691.69 724.86 810.78 866.71
463.52 576.21 777.17 906
0
150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900

S11


NEO-CY5-S4 497 (3.645) Cm (473:613)
320.04
100
%

640.55

342.84

648.37

137.04
670.80
141.04 671.69

969.78 1297.56
343.70 859.83
159.05 1298.51
968.87
368.18 392.47 435.27 860.79 961.79 1296.77
319.09 682.07 854.87 971.13 1012.55 1073.30
190.03 395.44 504.51 517.28 556.22 639.31 708.22 879.21 1088.461124.48 1282.89 1320.49 1356.92
234.57 453.52 738.47 795.40 912.09 142

150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200 1250 1300 1350 1400

NEO-CY5-S4

2.75e+1
3.68

2.5e+1

2.25e+1

2.0e+1

1.75e+1
1.14
1.5e+1
AU

Figure S6. Characterization of Boc-neomycin derivative and Neo-Cy5. a) LC-MS of 2-N-

1.25e+1 3.94

1.0e+1

trifluoroacetamidoethylthio-neomycin derivative. b) LC-MS of deprotected 2-N-

7.5

5.0

trifluoroacetamidoethylthio-neomycin
2.5

0.0
derivative. c) Analytical thin-layer
0.94

chromatography of the Neo-Cy5 conjugate. After selective deprotection of the 5” position

NEO-CY5-S4
-0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00 4.20 4.40 4.60 4.80 5.00 5.20 5.40 5.

of Boc-neomycin, the free amino group was reacted with Cy5 monofunctional NHS-ester 1.09
1.20
1.11 1.17
3.63 3.65

3.67

and the reaction monitored by TLC (silica gel 60 F254 plates; 1.24

chloroform/Ethanol/NH4OH 28%/5.6:3.8:0.45). The results showed that reactions were

3.68

1.28 3.71
5.41 5.6

complete. d) After purification on TLC plates (Silica gel 60, Merck), Neo-Cy5 was eluted
3.89 5.50
3.73
3.80 5.19 5.27 5.32
3.92
%

1.33

and the purity verified again by TLC. c) LC-MS characterization of Neo-Cy5. e) LC-MS of 1.37
1.41 3.02

the Neo-Cy5 conjugate (Cy5 from Lumiprobe, see Figure S5).

0.89 1.42
0.87 2.772.81 2.89 2.99 3.08 3.14 3.273.31 3.51
0.92 1.61
0.12 0.18 1.51 1.72 3.40 3.45
1.65 1.93
0.45 0.62 0.67 0.77
0.30 0.32

0.98

5
-0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00 4.20 4.40 4.60 4.80 5.00 5.20 5.40 5.
NEO-CY5-S4
3.76
3.73
3.72 3.79
0.00
3.81

3.59 3.67 3.90

3.92
4.00

S12
5

3.56

3.53

1.30
3.46
0.07 0.11 3.25 3.26 3.283.35
0.19 0.27 0.89 2.96 3.07
0.66 1.06 1.19 1.24 1.65 2.21 2.85 2.86
0.73
 90

80
Cy5 Ex
70
Neo-Cy5 Ex
Fluorescence (a.u.)

60

50 Cy5 Em
40
Neo-Cy5 Em
30

20

10

0
400
413
426
439
452
465
478
491
504
517
530
543
556
569
582
595
608
621
634
647
660
673
686
699
712
725
738
751
764
777
790
Wavelength (nm)

Figure S7. Excitation and emission spectra of Cy5 and Neo-Cy5. Data were recorded for
1 μM solutions of Cy5 or Neo-Cy5 in Tris-HCl (pH 7.6). Optical properties are identical.

Neomycin
4
3
Log 10 (survival percentage)

2
1
0
-1
-2
-3
-4
no antibiotic 0.75 0.85 1 1.5 Antibiotic µM
untreated 3,20 -1,06 -1,53 -2,14 -2,83
CCCP (30 µM) 3,10 2,21 1,97 2,08 1,23

Neo-Cy5
4
Log 10 (survival percentage)

3
2
1
0
-1
-2
-3
-4
-5
no antibiotic 32 48 Antibiotic µM
untreated 3,27 1,213199641 -3,863333333
CCCP (30 µM) 3,116 2,949488188 1,116666667

Figure S8. Neomycin and Neo-Cy5-induced cell killing is PMF dependent.
Representative experiments for survival of E. coli MG1655 cells after a 4.5-h treatment
with various concentrations of neomycin and Neo-Cy5 with and without a 2-h pre-
incubation with 30 μM CCCP. Plotted are values relative to untreated control culture.

S13

 10000

Fluorescence Neo-Cy5 (a. u.)

9000
8000
7000
6000
5000
4000
3000
2000
1000
0
0 1 2 3 4

Number wash
Figure S9. Excess Neo-Cy5 is removed by filtration. E. coli MG1655 cells (OD600 0.02)
were incubated 5 minutes with 32 µM Neo-Cy5 at 37 °C then filtered through a 0.45-µm
pore-size HAWP membrane. Cells were washed or not with 100 µL MOPS-G medium
sup Figure 1
(pre-warmed to 37 °C), recovered from the filter, and immediately analyzed by FACS.
Okuda et al., 2019
The average of Neo-Cy5 signals for each sample was measured. Three washing steps
were required to remove the Neo-Cy5.

Figure S10. Cy5 does not enter live E. coli cells. Left: Transmitted light image of a field of
E. coli MG1655 cells exposed to 32 µM Cy5 for 1 h at 37 °C. Cells were washed on a 0.45
µm-pore-size HAWP membrane filter. Right: Fluorescence image of the same field of
view obtained with an electron-multiplying gain of 50 and an exposure of 50 ms.

S14


Peripheral Neo-Cy5 and Spinach-ribosome
BF Cy5 Spinach Cy5 + Spinach

Figure S11. Neo-Cy5 does not localize with ribosomes after a 5-minute incubation. E.
coli cells expressing Spinach-tagged ribosomes were exposed to Neo-Cy5 and imaged.
Cells were illuminated with a 642-nm laser for Neo-Cy5 excitation and emission was
Sup fig 7
detected at 670 nm. Spinach aptamer bound to DFHBI was excited with a mercury lamp,
revised
and images were acquired with a GFP filter cube (ex. 470 ± 20 nm, em. 525 ± 25 nm).
Brightfield, Neo-Cy5 (red), Spinach (green), and merged images.

Fluorescence intensity (a. u.)

104
Fluorescence intensity (a. u.)

103 104

103

102 102 32 34 36 38
Time (s)

0 20 40 60 80 100 120
Time (s)
Figure S12. Fast kinetics of Neo-Cy5 interaction with the membranes revealed by real–
time FACS experiments. Cells in exponential growth phase (0.3 OD600) were diluted 20-
fold to a volume of 150 µL and placed into the flow cytometer for analysis at 37 °C. FACS
analysis was initiated and, after a delay of 20 s, 500 µL of Neo-Cy5 solution was added
for a final concentration of 0.4 µM. Addition of the drug diluted the cells to 0.003 OD600.
Fluorescence intensities of individual cells are plotted vs. time. Inset represents the
integration of the fluorescence signals within the boxed area. The signals were average
over 200-ms intervals during this 6-s time period.

S15



Figure S13: Neo-Cy5 induces morphological defects in E. coli cells grown overnight in
M9 glucose (0.4 % (w/v)). Bright-field images are displayed on the right and
fluorescence images on the left. At a concentration of 4 µM, cells are longer and at 8 µM,
foci are observed at poles.

S16

 BF TIRF TIRF (Cy5)
(Cy5) adjusted contrast

2 min

5 min

10 min

20 min

40 min

60 min

80 min

120 min Sup F

Figure S14: Time-course experiment of changes in Neo-Cy5 accumulation levels. Fast
growing cells (OD600: 0.02) were incubated at 37 °C with Neo-Cy5 (32 µM). At each time
points cells were washed on filter and then observe directly on MOPS-G agarose pad
(Scale bar: 5 µm). Images were scaled to the minimum and maximum pixel values of the
first image t=2mn. For the first 4 time points, contrast was adjusted for better
visualization and displayed on the right column. For each time point, images were
analyzed with microbeJ for quantification of Cy5 signals to generate figure 5 panel d.

S17